A Comprehensive Guide to Understanding Influences on PCR Reactions

PCR (Polymerase Chain Reaction) stands as a cornerstone technique in molecular biology, empowering a wide array of applications ranging from gene detection to pathogen identification. Yet, the efficacy of PCR assays can be compromised by inhibitory factors that lurk within the reaction milieu. These inhibitory factors may emanate from both internal components and external contaminants, posing challenges to the accuracy and reliability of PCR results.

Internal Factors in PCR Reactions

1. Primer Design:

PCR primers, synthesized custom DNA oligonucleotides, play a pivotal role in determining the specificity and efficiency of PCR amplification. The success of PCR largely depends on adhering to certain principles in primer design.

• Optimal Primer Length: Primers typically range from 15 to 30 nucleotides in length. Excessively long primers hinder binding to the template and may form secondary structures, while overly short ones compromise specificity.
• Appropriate GC Content: The GC content of primers should ideally fall within the range of 40% to 60%. Consistency in GC content between forward and reverse primers is crucial for balanced annealing. Extreme GC content can impede primer-template annealing. The bases at the 3' end of the primer generally avoid A/T pairs because A/T pairs have only 2 hydrogen bonds (compared to 3 in G/C pairs), which relatively increases the efficiency of mispriming. Additionally, the 3' end should prevent consecutive C or G bases because continuous GC pairs are detrimental to the binding of the primer's 3' end with the template.
• Avoidance of Complementary Sequences: Primers must not contain complementary sequences to each other or within themselves. Random distribution of bases is essential to prevent primer dimer formation, which can interfere with template annealing.
• Consideration of Terminal Modifications: While modifications at the 5' end of primers can enhance specificity, modifications at the 3' end should be avoided as they can interfere with extension. Modifications at the 5' end include enzyme cleavage sites, fluorescent or biotin labels, introduction of point mutations, insertions, deletions, or promoter sequences.
• Optimal Tm Values: The melting temperature (Tm) of primer-template pairs should ideally fall between 55°C to 80°C. Consistency in Tm values between forward and reverse primers ensures balanced annealing, typically not exceeding a 5°C difference.
• Prevention of Secondary Structures: Primers should be designed from genomic regions less prone to forming secondary structures to avoid hindrance in amplification.
• Avoidance of Terminator Codons: Terminal primer bases should not correspond to terminator codons (UAA, UAG, UGA) as the third position of these codons is prone to degeneracy, affecting amplification specificity and efficiency.

2. Primer quality: It's essential to ensure that the oligos are synthesized with a high coupling efficiency, ideally around 99%. Purification methods such as RPC, PAGE, or HPLC can further enhance the quality by removing impurities. High coupling efficiency allows for the synthesis of longer oligos, up to 200 bases, while modifications can be tailored to specific research needs. Additionally, analyzing oligos through PAGE and employing techniques like Maldi-Tof Mass QC can provide assurance of their quality and reliability for PCR applications. These measures collectively contribute to obtaining accurate and reproducible results in molecular biology experiments.

3. Enzyme and its concentration: Excessive enzyme concentration can lead to nonspecific amplification, while too low a concentration reduces the synthesis of products.

4. Quality and concentration of dNTPs: The dNTP solution is typically acidic, with adjustments made to achieve a pH of 7.0-7.5 during system preparation. Ideally, under standard conditions, the concentrations of all four dNTPs should be equal. Deviations from this balance can lead to issues such as mispriming if one dNTP is excessively high, or reduced PCR product yield if concentrations are too low. To mitigate these risks, we recommend opting for a ready-to-use dNTPs mix solution containing dATP, dCTP, dGTP, and dTTP (monosodium salts) at an identical concentration, dissolved in sterile deionized water at pH 7.5. This solution should exhibit a purity of ≥99% as determined by High-Performance Liquid Chromatography (HPLC) analysis, and importantly, it should be free of RNase and DNase contaminants.

5. Nucleic acid template: The quantity, purity, and integrity of the template all affect the PCR reaction.

6. Mg2+ concentration: Mainly affects the specificity and yield of PCR amplification, generally corresponding to the concentration of dNTPs. An excessively high Mg2+ concentration reduces specificity, leading to nonspecific amplification, while too low a concentration decreases the activity of Taq DNA polymerase, resulting in reduced product yield.

7. Temperature and time settings: PCR involves three temperature points—denaturation, annealing, and extension—each with its own time setting. For templates with high GC content, the denaturation time should be extended. If the primer has a low number of bases, annealing temperature can be raised slightly to increase PCR specificity; conversely, if the primer has a high number of bases, annealing temperature can be lowered to facilitate primer binding to the DNA template. For longer product lengths, extension time should be extended accordingly.

8. Number of cycles: The number of cycles determines the extent of PCR amplification, primarily dependent on the concentration of template DNA. The more cycles, the higher the amount of nonspecific products and the higher the rate of base misincorporation.

External Factors in PCR Reactions

• SDS (Sodium Dodecyl Sulfate): Anionic detergent that can disrupt the non-covalent bonds (hydrogen bonds and hydrophobic interactions) of enzyme proteins, binding to the hydrophobic regions of proteins, thereby denaturing enzyme proteins and causing loss of their natural conformation and function. A concentration of 0.01% can completely inhibit PCR reactions, while 0.005% can significantly reduce yield.
• Phenol: Organic solvent capable of denaturing enzyme proteins. A concentration of 0.5% can completely inhibit PCR reactions, while 0.2% can significantly reduce yield.
• Ethanol: Organic solvent; concentrations greater than 1% can inhibit PCR reactions.
• Isopropanol: Organic solvent slightly stronger in inhibiting PCR reactions compared to ethanol.
• Sodium Acetate (NaAc): Inhibits PCR reactions at concentrations greater than 5mM. Causes a decrease in reaction system pH, forming sodium salt complexes with DNA, leading to precipitation.
• Sodium Chloride: Inhibits PCR reactions at concentrations greater than 25mM.
• EDTA: Metal ion chelator capable of chelating Mg2+ in the system, inhibiting polymerase activity. A concentration of 0.5mM reduces PCR product yield, while 1mM completely abolishes PCR product.
• Hematin: Hematin is a heme pigment found in higher animals, serving as the prosthetic group of hemoglobin, present in the color proteins of the blood, liver, and muscles of higher animals. Inhibits PCR reactions at concentrations greater than 1mg/ml. Difficult to remove with organic solvent extraction of DNA, leading to easy residue.
• Hemin Chloride: Hemin chloride is hematin's central ion, divalent iron ion, which, in the presence of oxidants and heating, can be oxidized to trivalent iron ion, forming brown hemin chloride. Inhibits PCR reactions at concentrations greater than 0.1ng/μl.
• Tannic Acid: Tannic acid, also known as tannin acid, is a natural phenolic organic compound found in plants. Inhibits PCR reactions at concentrations greater than 0.1ng/μl.
• Heparin: Inhibits PCR reactions at concentrations greater than 0.15 IU/ml. Heparin, composed of sulfated D-glucosamine and D-glucuronic acid, is a viscous polysaccharide containing various sulfate groups, exhibiting strong acidity. Heparin, associated with its anions, is related to anticoagulation and thus is a polyionic glycosaminoglycan drug. It also exists in tissues such as the lungs, vascular walls, and intestinal mucosa, serving as a natural anticoagulant in animals. Heparin strongly inhibits MLV reverse transcriptase and Taq DNA polymerase. During nucleic acid purification, heparin in the sample can bind to DNA and RNA, and subsequent steps cannot remove this interfering effect of heparin.
• Urea: Derived from urine, urea is an organic compound composed of carbon, nitrogen, oxygen, and hydrogen. Inhibits PCR at concentrations greater than 20mM. Urea is the final product of protein metabolism in the body, primarily excreted via filtration by the kidneys into urine.
• Humic Acid: Derived from soil, plant materials, and natural water sources. It may inhibit polymerase activity through chelation of Mg2+. Humic acid is an organic substance formed by microbial decomposition of dead matter in the soil, typically black-brown in color, containing elements necessary for plant growth and development, thus improving fertility. It constitutes a major part of soil organic matter, typically accounting for 50-70% of total organic matter. Major elements in humic acid include carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, etc. Humic acid is not a single compound but rather a mixture of organic compounds with commonalities and differences in composition, structure, and properties, primarily consisting of humic and fulvic acids.
• Bile Salts: Found in feces. Bile salts, also known as bile acids, are amphipathic macromolecules derived from cholesterol in bile, aiding in the digestion and absorption of cholesterol and fats, leading to elevated blood lipid levels. Bile salts effectively break down fat tissues and proteins, facilitating the faster breakdown, absorption, and utilization of these nutrients in the small intestine.
• Bilirubin: Bilirubin is a type of bile pigment, the primary pigment in human bile. It is the major metabolic product of heme compounds in the body, toxic and capable of irreversible damage to the brain and nervous system, yet it also functions as an antioxidant, inhibiting the oxidation of linoleic acid and phospholipids. Bilirubin serves as a critical indicator of jaundice and liver function clinically. Bilirubin can inhibit Taq enzyme activity.
• Hemoglobin: Hemoglobin and its metabolites may interact with Taq enzyme, inhibiting Taq enzyme activity and significantly reducing PCR amplification efficiency. This inhibitory effect may be related to the release of iron ions.
• Hyperlipidemia: High levels of lipids inhibit Taq enzyme activity, resulting in reduced amplification efficiency. The interference mechanism of hyperlipidemia in Real-Time PCR mainly involves the shielding or absorption of fluorescence by low-density lipoproteins in the blood. Hyperlipidemia leads to fluorescence quenching, resulting in decreased fluorescence signal intensity. Studies have shown that the use of low-temperature high-speed centrifugation can effectively remove interference from lipemic factors.
• IgG: The mechanism by which IgG inhibits PCR reactions involves interaction with single-stranded DNA, preventing target DNA from binding to DNA polymerase. Moreover, this inhibitory effect is enhanced during sample heating cycles.
• Sodium cholate, deoxycholate, taurocholate, iron chloride, plant complex polysaccharides, melanin, calcium ions, collagen, etc.

PCR Enhancers

PCR enhancers are a type of additive that can improve the sensitivity, specificity, fidelity, and other properties of PCR and PCR-related techniques. Examples include betaine, dimethyl sulfoxide (DMSO), formamide, glycerol, PEG, spermine, and single-stranded DNA binding proteins. However, at high concentrations, they can also inhibit PCR. According to their functions and principles, they can be roughly divided into three categories:

1. Used for amplifying templates with high GC content or complex secondary structures: 1M betaine, 1-10% DMSO, 1-5% formamide, and 1-10% glycerol can significantly increase the yield for such templates, but excess amounts can inhibit PCR reactions.

• Betaine: Betaine, chemically known as N,N,N-trimethylglycine, is a biogenic amine similar in structure to amino acids and belongs to the quaternary ammonium compound category. Betaine is widely present in both animals and plants. Betaine has the ability to disrupt DNA sequences rich in GC content, improving the yield and specificity of PCR amplification.
• Dimethyl Sulfoxide (DMSO): DMSO is a sulfur-containing organic compound with weak basicity, soluble in both water and organic solvents, commonly used as an organic solvent. Pure DMSO has a freezing point of 18.45°C, while DMSO with 40% water content does not freeze at -60°C, and when mixed with water or snow, DMSO generates heat. Therefore, it provides convenience for making antifreeze for automobiles, brake fluid, and hydraulic fluid components.

2. Used to protect the activity and stability of DNA polymerase

In some reactions, additives such as 0.1mg/ml BSA, 0.1-10% gelatin, or non-ionic detergents can enhance the stability of polymerase and reduce adsorption of reagents to tube walls.

BSA can resist the inhibitory effects of hemoglobin, melanin, etc. Non-ionic detergents such as Tween-20, NP-40, TritonX-100 can counteract the inhibitory effects of trace amounts of strong ionic detergents, such as 0.01% SDS.

• Trehalose: Trehalose, also known as mycose, is a non-reducing disaccharide composed of two glucose molecules. It forms a special protective membrane on the surface of cells under harsh conditions such as high temperature, extreme cold, or dehydration, effectively protecting the structure of biomolecules and maintaining the life processes and biological characteristics of organisms. Trehalose has thermal stability and heat-activation properties. It can maintain the protein skeleton during protein denaturation and preserve its natural structure. It has been verified in various enzymatic reactions in molecular biology, such as cDNA synthesis involving reverse transcriptase, DNA degradation involving Dnase I, and restriction enzyme cleavage reactions.
• Functions:
• Acts as a PCR enhancer by lowering the DNA double-stranded melting temperature, maintaining the stability of Taq DNA polymerase, and improving PCR reaction efficiency.
• Neutralizes the inhibitory effects of inhibitors on DNA polymerase, maintaining PCR amplification efficiency.
• Acts as an additive in SYBR Green I real-time fluorescence quantitative PCR, effectively reducing the inhibition of SYBR Green I on PCR.
• Acts as a preservative for rare sample genomes or genomes at very low concentrations.
• Applications:
• Acts as an enzyme stabilizer, maintaining enzyme activity and post-heat activation enzyme activity.
• Can be applied in DNA sequencing reaction experiments to enhance the accuracy of sequencing containing repetitive sequences (multiple consecutive A's or T's).
• PCR amplification of complex templates, such as samples with high GC content.
• Suitable for reverse transcription reactions of relatively long cDNA, especially the synthesis of full-length cDNA, to improve library coverage.
• Preservative for trace DNA samples.
• Additive for qRT-PCR reaction systems to improve reaction performance.
• BSA and Tween-20: Trehalose, BSA, and Tween-20 have anti-inhibitory effects on shrimp shell components and are commonly used in PCR methods for detecting viruses in shrimp samples. Additionally, BSA can enhance the resistance of rTth enzyme to biological samples, and BSA has a high lysine content, which can bind to phenolic compounds remaining in the nucleic acid extraction process, protecting Taq DNA polymerase activity and thereby enhancing PCR amplification efficiency.
• PEG600 and Glycerol: PEG600 and glycerol can increase PCR product yield by enhancing enzyme thermal stability and protecting enzyme activity.

3. Used to optimize primer and template binding

The addition of ammonium ions can reduce mismatches between primers and templates, improving reaction specificity, thus reducing the requirements for reaction conditions. Therefore, many PCR reagents contain 10-20mM ammonium sulfate. Tetramethylammonium chloride (TMAC) also plays a similar role.

• Spermine: Spermine, also known as tris(aminopropyl)amine hydrochloride, is a polyamine. It is widely distributed in organisms and is biosynthesized from putrescine (butanediamine) and S-adenosylmethionine. Spermine can inhibit neuronal synthetase, bind and precipitate DNA, be used for DNA-binding protein purification, and stimulate T4 polynucleotide kinase activity. Research has shown that spermine exhibits significant inhibitory effects on bile salts, urea, and hematin. Spermine enhances PCR, primarily by promoting the formation of PCR initiation complexes through promoting the binding of polymerase, primers, and templates. Micromolar concentrations of spermine significantly enhance the amplification of plant genomic DNA.
• Ammonium Sulfate: Ammonium sulfate can optimize the specific binding of primers to templates, improving PCR specificity. Research results show that ammonium sulfate has a significant anti-urea inhibitory effect.

4. Sources and Mechanisms of PCR Inhibitors in Specimens

• Endogenous Sources: Naturally occurring components present in specimens, such as immunoglobulins, proteases, hemoglobin and its metabolites, lactoferrin in leukocytes, myoglobin, lipids, mucins, ions, bile salts, polysaccharides, etc.
• Exogenous Sources: Inhibitors present in specimen containers or sampling equipment, such as heparin anticoagulants, glove powder, etc.

1. Mechanisms of Inhibition:

• Interference with cell lysis during nucleic acid extraction: Incomplete cell lysis leads to inefficient release of nucleic acids, preventing amplification. For instance, DNA released by boiling methods may not always fully dissociate from structural or DNA-binding proteins, resulting in inhibitory effects on amplification.
• Degradation or encapsulation of nucleic acids: DNA instability at the primary structure level due to hydrolysis, non-enzymatic methylation, oxidative damage, and enzymatic degradation. Nucleases present in cells can degrade nucleic acids. Residual nucleases in nucleic acid samples can lead to nucleic acid degradation. Restriction endonucleases produced by microbes are also factors in DNA degradation. Some bacterial DNAases are heat-stable nucleases, which can hydrolyze genomic and primer DNA during mid-amplification. Nucleic acids and primers may fail to amplify due to their inability to bind to DNA polymerase. Inhibitory effects of proteins, polysaccharides, cell debris, lipids, etc., on PCR are likely due to their physical encapsulation of DNA, preventing its contact with the polymerase. The impact of talcum powder on PCR amplification on latex gloves may be due to its non-specific binding to DNA. Because DNA can bind to glass, silica, etc., this is also the basic principle of nucleic acid extraction using silica adsorption methods.
• Inactivation of heat-stable DNA polymerase.

2. Clinical PCR inhibitors: total bilirubin, hemoglobin, IgG, triglycerides, and heparin. These substances originate from sample components or processes and can reduce the efficiency of template amplification, leading to false-negative results in the detection of low-level samples.

3. Reverse transcription inhibitors: SDS, EDTA, glycerol, sodium pyrophosphate, spermine, and guanidinium salts. Washing RNA precipitates with 70% (v/v) ethanol can remove inhibitors.

In conclusion, understanding and controlling both internal and external factors influencing PCR reactions are crucial for obtaining reliable results in molecular biology experiments. By adhering to principles of primer design, ensuring primer and enzyme quality, optimizing reaction conditions, and mitigating the effects of inhibitors, researchers can enhance the accuracy and efficiency of PCR-based assays. At SBS Genetech, we are committed to providing high-quality products and solutions that contribute to the success of PCR and other molecular biology applications. With our reliable reagents and expertise, researchers can confidently tackle complex scientific challenges and drive forward innovations in the field.